1. Field of the Invention
The present invention relates to methods of increasing protein expression levels whereby at least one amino acid in a polypeptide amino acid sequence is substituted for the amino acid, proline.
2. Background
The ability to obtain a high level of expression of secreted recombinant proteins in yeast and mammalian cells is often found to be protein dependent. Efforts in maximizing recombinant protein expression are often focused on increasing the levels of the mRNA of the recombinant gene. However, the rate-limiting step in the expression of certain proteins is not the level of mRNA but rather is due to inefficiencies in the folding, addition of post-translational modifications and secretion of the recombinant proteins.
Combining random mutagenesis with the display of proteins on the surface of yeast is a powerful technique to identify proteins with altered properties. In this technique, mutant proteins are displayed on the cell surface and screened for either higher-affinity binding or increased expression levels of the displayed protein by FACS (fluorescence activated cell sorting). Display of mutant libraries on the yeast cell surface has been used to identify mutants of both single-chain antibodies and single-chain T-cell receptors that have a higher affinity for antigen or peptide/MHC, respectively. The yeast surface display method has also been used to improve the level of expression of a single-chain T-cell receptor.
There remains a need in the art for identifying structural characteristics or features of proteins which permit high expression of recombinant proteins, particularly eukaryotic proteins, whose expression may be limited due to inefficiences in folding, addition of post-translational modifications and/or secretion of proteins.